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       【摘要】 
       ObjectiveThis research intended to study and identify the antibacterial activity of Lo Han Kuo fruit. MethodsThe fruit extract was prepared from dry Lo Han Kuo fruit and then screened for antibacterial activity using a colorimetric detection method. To further isolate the bioactive fractions, the fruit extract was separated into smaller fractions via high-performance liquid chromatography (HPLC) and the fractions were tested again for antibacterial activity. The positive fractions showing antibacterial activity were further tested by blood agar plating method.ResultsLo Han Kuo fruit extract exhibited strong antibacterial activity against the oral bacteria, S. mutans. After seperated by HPLC, two major HPLC fractions of Lo Han Kuo fruit extract, fraction 18～19 and 34～35, demonstrated significant inhibition for oral bacteria S. mutans, while other fractions exhibited some, but not complete inhibition. Furthermore, the antibacterial activity seems to be not related with mogrosides because the pure mogroside V exhibited no antibacterial activity.ConclusionThis is the first study demonstrating that Lo Han Kuo fruit extract exhibited strong antibacterial activity but not due to the presence of mogrosides.
       【关键词】  Momordica grosvenori HPLC fractionation Antibacterial activity Mogrosides
       1  Introduction
       Dental caries is the predominant cause of tooth loss in children and young adults. Caries is a bacterial infection caused by specific bacteria, albeit, this is not limited to a monospecific pathogen ［1,2］.  The presence of "mutans streptococci" and lactobacilli has been associated with caries development.  Although treating oral bacteria with conventional antibiotics is an option, plant-based "antibiotics" products could be important alternatives to the traditional antibiotics to combat the recent development of antibiotics-resistant bacterial strains due to the overuse of over-the-counter antibiotics ［3～5］.  Plant materials and plant parts have been used as medicine in China to treat diseases for centuries.  Plants are the rich source for drug discovery due to the secondary metabolites produced.  Plant-based "antibiotics" products can serve as an important source for future therapeutic treatment of infectious diseases.
       
       Lo Han Kuo (Momordica grosvenori) has been used in China for centuries as sweetening agent ［6］.  Lo Han Kuo fruit is also used to treat sore throat and coughing for many years. However, very little critical scientific research has been conducted to evaluate the potential therapeutic functions of Lo Han Kuo components. Some studies reported that the sweetening agent mogrosides are the principal components for the antibacterial activity of Lo Han Kuo fruit, especially mogroside V ［7～9］.  But those studies either lack of good bioassays or pure Lo Han Kuo fruit extract samples.  Those studies were incomplete because only fruit extract or partially purified mogrosides were used.
       
       This study critically evaluated the anti-bacterial activity of Lo Han Kuo using HPLC fractions and pure mogroside V compound.  HPLC fractionation was performed to isolate fruit extract into fractions, and the bioactivities of these fractions were tested with functional bioassays.  Our experiment data demonstrated that the LHK extract not only had the intensive sweetening capacity, but might also have potential health benefits, including inhibiting the growth of Streptococcus mutans that would be predicted to reflect an anti-caries activity. 
       2  Materials and Methods
       2.1  Bacterial strain and growth conditionOral bacteria was used in this study. Streptococcus mutans (ATCC 25175) was purchased from ATCC.  TSBYE media and Anaerobe Broth were purchased from Oxoid Ltd. Growth conditions were at 37℃ in anaerobic condition.
       2.2  InstrumentsHPLC column: Phenomenex Gemini 5μl C18, System: Waters 510 pumps ［2］, Waters 484 Tunable Absorbance Detector; Microplate reader: MRX plate reader (Dynatech Laboratories). Evaporation equipment: Savant SC-110 Speed Vac, Savant RT-100 Refrigerated Condensation with a Savant VP 100 oil pump; water purification equipment: Milli-Q;
       2.3  ChemicalsMethanol, resazurin and ethanol were purchased from Sigma, USA. Blood agar plates were obtained from Remel Company, USA.
       2.4  Lo Han Kuo extractDry Lo Han Kuo fruit was purchased from The Luohanguo Company of Chinese Guangxi Yongfu. Lo Han Kuo fruit extract was prepared from Lo Han Kuo fruit by crushing 10 grams of dry Lo Han Kuo fruit using a mortar and pestle and followed by adding 20 ml of 50% EtOH.  Lo Han Kuo extract was allowed to shake for 24 hours in a flask being filtered through filter paper. One ml of filtered extract was then lyophilized 8 hours into dry powder. The dried extract obtained through the lyophilization process was re-suspended in 100μl of 50% EtOH.
       2.5  Cell density measurement of S. mutans cultureThe effect of LHK extract on the growth of S. mutans was examined by measuring the cell density at OD600nm of the cultures in TSBYE media, and was compared to growth in an equivalent concentration of sucrose in TSBYE media.  Briefly, overnight bacterial culture was diluted 10 times in fresh culture media.  Various concentrations of LHK extract (10, 5, 0 mg/ml) were suspended in the TSBYE media.  500 μl of an S. mutans overnight culture was inoculated into culture tubes containing 5 ml of the LHK solutions and incubated at 37℃ in an anaerobic chamber for 24 hr.  Sucrose in TSBYE media and TSBYE media alone were used as positive and negative controls respectively.  At 24 hr, the OD600nm of the culture solutions were measured.
       2.6  HPLC purification procedure300 ml of the LHK extract (165mg/ml, dissolved in water) was filtered with ULTRAFREE-MC 0.45 μm Filter Unit and was injected using Waters 717 plus Autosampler to the Waters 510 HPLC system (contains Waters 510 HPLC Pump, Waters 484 Tunable Absorbance Detector, Waters System Interface Module and Millennum 32 software). An RP18 7 μm, 19 mm×150 mm Waters Symmetry Column was used.  Flow rate: 1.0 ml/min.  With a step gradient using mixtures of methanol/water, fifty fractions were collected with (4 ml per fraction) Spectra/Chrom CF-1 Fraction Collector.  All fractions were dried under room temperature using evaporating equipment (contains Savant SC-110 Speed Vac, Savant RT-100 Refrigerated Condensation and Savant VP 100 oil pump).
       2.7  Bioassay screening of Lo Han Kuo fractionsEach fraction (1 ml) from the HPLC was lyophilized and tested against S. mutans using the high throughput screening(HTS) method(10). Lyophilized extracts were dissolved in 50 μl of 50% EtOH and tested for activity through the HTS procedure by adding 1 μl of each extract to selected wells of a 96-well plate containing 100μl of TSBYE medium and 10% of bacteria from the overnight culture. The plate was then incubated in an anaerobic chamber between 16 and 18 hours. After incubation, 3μl of the colorimetric indicator resazurin was added to each well. The live bacteria could metabolize the blue resazurin into pink color, while the dead bacteria could not. The plate was then allowed to incubate for the resazurin to be metabolized by the bacteria providing the change in color. Wells containing the S. mutans required 1 hr of incubation.  A reading using a Dynex 96 well plate reader is taken for each bacteria at 600nm.  Typically wells with viable cell will range in pink color giving an OD595 reading of 0.200 to 0.600.  Wells with nonviable bacteria will be blue giving an OD595 reading above 2.000.
       2.8  Blood agar platingFractions with blue colors in the 96-well plate that showed biological activity via the HTS were diluted 10,000x and plated in duplicate on blood agar plates (Remel).  Controls were also plated along with wells that did not show activity around the active ones (e.g. If fraction 18 showed activity we would plated fractions 17, 18, and 19.)  The plates were incubated at 37℃ in an anaerobic condition for 48 hours.  The colonies (CFU) were counted on each agar plate after the incubation.
       3  Results
       The effect of LHK extract on the growth of S. mutans was examined.  LHK fruit extract demonstrated a dose-response inhibition of S. mutans growth (Fig. 1) when compared to the control cultures of sucrose in TSBYE media in overnight culture. The LHK extract reduced the density of S. mutans.  LHK concentration of 10mg/ml significantly reduced the S. mutans density by 30%.
       Preparative HPLC of Lo Han Kuo fruit extract was performed and fifty fractions were collected (Fig. 2).  Fig. 3 displays the profile of this separation that was subsequently used to examine biological functions of the extract fractions.
       
       Bioassay for HPLC fractions of Lo Han Kuo extract was conducted in 96-well microplates (350 μl well volume) in an anaerobic chamber at 37℃.  Briefly, 20 μl of S. mutans overnight culture was added to each well containing various concentrations of sucrose.  The OD of each well was measured at baseline.  Individual HPLC fractions (5 μl) were added to each well and incubated overnight.  Resazurin was then added to each well to detect metabolic activity related to the amount of growth that had occurred.  The results, Fig. 3, demonstrate significant inhibitory activity of fraction collected at 19 and 35 min.  Fig.4 showed the antibacterial profiles of the fifty fractions of HPLC.  Blood agar plates were used to further confirm the antimicrobial properties of HPLC fractions.  The CFU on blood agar plates were counted and recorded.  HPLC Fraction 18, 19 and 34, 35 had the fewest CFUs.Fig. 5 shows an example of LHK fraction #35 that can significantly inhibit the growth of S. mutans.  Compared to the control, LHK fraction #35 shows the bacteriastatic activity.Purified mogroside V exhibited no inhibition against either oral bacteria, A. actinomycetemcomitans and Streptococcus mutans (Fig. 6). 
       4  Discussion
       Lo Han Kuo extract exhibited antibacterial activity against S. mutans.  Lo Han Kuo fruit extract was further purified with HPLC.  The bioactivities were identified. The antibacterial activities of Lo Han Kuo fruits were identified in two major fractions.  The fractions 18～19 are more polar than fractions 34～35.  Current research work is focused in isolating and identifying the bioactive compounds in those fractions.
       Surprisingly, the mogroside V did not show any antibacterial activity against oral bacteria.  Our study indicates the antibacterial activity does not come from mogrosides. This experimental data raised the possibility that the studies on mogrosides of Lo Han Kuo extract by others were incomplete because they used Lo Han Kuo extract instead of the pure mogrosides ［7,8］.  Even though the mogrosides are the main components of Lo Han Kuo fruits, it does not mean the antibacterial bioactivities come from mogrosides as other components could be responsible for the antibacterial activity in Lo Han Kuo fruit.
       
       This is the first study that demonstrated mogroside V had no antibacterial activity.  Whether the other mogrosides, such as mogroside I to IV, have any antibacterial activity are still to be determined.  It is very likely other chemical components in Lo Han Kuo fruit are responsible for the antibacterial activity.  Future research work will be focused in isolating and identifying the bioactive compounds from Lo Han Kuo fruit.
       
       Dental caries is the predominant cause of tooth loss in children and young adults.  Caries is a bacterial infection caused by specific bacteria, albeit, this is not limited to a monospecific pathogen. Plant-based "antibiotics" products from Lo Han Kuo fruit could be important alternatives to the traditional antibiotics to combat the recent development of antibiotics-resistant bacterial strains due to the overuse of over-the-counter antibiotics.  Lo Han Kuo fruits have been used as medicine in China to treat sore throat and Strep bacterial infection for centuries.  The antibacterial components isolated from Lo Han Kuo fruits can serve as an important source for future therapeutic treatment of oral diseases. All wells contain: blue color no metabolic activity of live bacteria, pink color positive for bacterial growth and metabolism. The dark purple color well is for HPLC fraction #19 and the blue color well is for HPLC fraction #35.
       10 μl of a control well without LHK fraction (Left) or blue well with LHK fraction #35 (Right) was diluted to 10-4 and 50 μl was distributed onto a blood agar plate. The left plate contained 10 times more S. mutans CFUs than the right plate.
       Acknowledements:This research was supported by the grant R41DE17265-01 from the NIH/NIDCR, U. S. A. We would like to thank Dr. Marshal, McGill University, Canada for providing us the purified mogroside V sample. CFUs of control without LHK fraction (Left) or pure mogroside V (Middle) or fraction #35 (Right). Control and mogroside plates contained 10 times more S. mutans than the fraction#35 plate.
       【参考文献】
         　　［1］Allukian M, Jr. The neglected epidemic and the surgeon general"s report: A call to action for better health［J］.AJPH 2000，90:843.
       
       ［2］Milgrom, P, Weintstein, P. Early Childhood Caries: A team approach to prevention and treatment［J］.University of Washington, Seattle. 148p.
       
       ［3］Kuzma L, Rozalski M, Walencka E, Rozalska B, Wysokinska H. Antimicrobial activity of diterpenoids from hairy roots of Salvia sclarea L.: Salvipisone as a potential anti-biofilm agent active against antibiotic resistant Staphylococci［J］.Phytomedicine. 2007，14(1):31-5. Epub 2005 Nov 15.
       
       ［4］Ball AR, Casadei G, Samosorn S, Bremner JB, Ausubel FM, Moy TI, Lewis K. Conjugating berberine to a multidrug efflux pump inhibitor creates an effective antimicrobial［J］.ACS Chem Biol. 2006，1(9):594.
       
       ［5］Francolini I, Norris P, Piozzi A, Donelli G, Stoodley P. Usnic acid, a natural antimicrobial agent able to inhibit bacterial biofilm formation on polymer surfaces［J］.Antimicrob Agents Chemother. 2004，48(11):4360.
       
       ［6］Lee C. Intense sweetener from Lo Han Kuo (Momordica grosvenori)［J］.Experientia. 1975，31(5):533.
       
       ［7］穆静.罗汉果浸出液对变链菌致龋作用的实验室研究［J］.中华口腔医学杂志， 1998，33（3）：183.
       
       ［8］Zhou JH. Composition and method for inhibiting oral bacteria［J］.US Patent 6,319,523. June 29, 2000.
       
       ［9］黄志江,黄捷,孙滢川,王霆,刘国卿. 罗汉果的药用研究［J］.广西师范大学学报(自然科学版). 1998, 16（4）:75.
       
       ［10］Ying Zhou, C.B. Huang, Identification of the Antibacterial Activity of Morus rubra Leaves by a High-Throughput Screening Method［J］.时珍国医国药，2007，8: in press.